We are studying the effect of activation upon synapse structure and function during development in vitro and in vivo. Activity-dependent synapse elimination is a major process determining nervous system performance and we present evidence that receptors or other molecules at the synapse are acted on by different protein kinases as a critical step in this process. We seek to extend our studies to some neurodevelopmental disorders such as autism. 1) Activity-dependent synapse loss and stabilization at the neuromuscular junction (Nelson, Jia, Li, Yang Lanuza, Valoria, Gizaw). We have utilized a compartmental tissue culture system (Fig. 1) to analyze the cellular locus of some of the processes involved in activity-dependent synapse modulation. Two physically separate populations of cholinergic neurons (in the side compartments of the system) converge on single population of muscle fibers. A subpopulation of these muscle fibers become innervated by axons from both of the neuronal populations. Stimulation of one set of axons produces down regulation of the synaptic efficacy of the synapses from the other, non-stimulated neuronal population in those muscle cells innervated by both populations. Previous work had shown that Protein Kinase C (PKC) action was required for expression of this Hebbian, activity-dependent synapse modification. This work involved pharmacological activation and blockade of PKC and the use of cells from animals in which the PKC theta isoform had been knocked out. Since the muscle cells and neurons are prepared and plated separately, we were able to assess the effect of selective loss of PKC from neurons or muscle cells by using heterologous preparations in which either nerve or muscle lacked the PKC while the other cell type was normal. We had evidence that the down-regulation of synapse efficacy involved post-synaptic changes including loss of acetylcholine receptors (AChR). As we therefore expected, when normal nerve innervated muscle lacking PKC theta, we found no activity-dependent synapse modulation. More surprisingly, the modulation was also not expressed when PKC theta deficient nerve innervated normal muscle. This was true with regulation induced by either electrical stimulation or PKC activation by application of the phorbol ester PMA. Furthermore, this effect of neuronal PKC theta deficiency was itself activity dependent. In such preparations that were lacking PKC theta in the neuronal component and that were electrically silenced with the Na+ channel blocker, tetrodotoxin, PMA did produce synapse down regulation. We interpret these rather complex findings, and other findings with Protein Kinase A (PKA) agonists and antagonists, in terms of the model shown in Fig. 2. PKA and PKC play antagonistic roles in both pre- and post-synaptic components of the synapse. PKA actions are necessary to sustain transmitter output from the nerve terminal and to stabilize the postsynaptic receptor. PKC, by contrast, blocks release of receptor stabilizing material, perhaps a peptide (such as Calcitonin Gene Related Peptide or CGRP) from the nerve and acts in the muscle to de-stabilize the AChR. Considerable evidence from the literature suggest that this phosphorylation model may underlie some examples of activity-dependent synaptic plasticity in the central nervous system.2) Autocrine function for GDNF at the neuromuscular junction (Yang, Nelson) Glia Derived Neurotrophic Factor (GDNF) is known to be a trophin produced by muscle with powerful effects on spinal motor neurons and other nerve cells throughout the brain. We have examined the possibility that GDNF could also have an effect on the muscle cell itself, in particular on the metabolism and membrane localization of the acetylcholine receptror (AChR). We find that GDNF does increase the insertion rate of AChR into the surface membrane of the muscle cells, with a lesser effect on receptor loss from the membrane and without having any appreciable effect on receptor synthesis. A number of inhibitor studies suggest that the GDNF effect is mediated by the alpha 1 GDNF receptor and involves the Ret receptor, MAP kinase, cAMP/CREB and Src kinase activity. Thus, this trophin may act in a synergistic pre-and postsynaptic manner to modify synapse efficacy. 3) Molecular basis for neurodevelopomental disorders (Nelson, Satyanarayana, Song, Kuddo, VanDunk, in collaboration with J. Grether and K.B. Nelson) We have access to archived neonatal blood spots from normal children and children subsequently found to be autistic. The small volumes of the available samples, the low levels of some of the analytes of interest and the high levels of proteins including hemoglobin in the material eluted from the blood spots have posed considerable technical problems. We find that high levels of materials that interfere with the assay for Brain Derived Neurotrophic Factor (BDNF) are present in the blood eluates. With a combination of sample dilution with an assay buffer and the use of known analyte spikes we show that samples from autistic and control children do not differ either in the the levels of BDNF or in the levels of the interfering substances. Similarly, levels of IL-1, IL-4, IL-8, TNF alpha and VEGF do not distinguish cases and controls in our sample. Collaborators Judy Grether, Ph.D. California Department of Health Services, Berkeley, California Karin B. Nelson, M.D., Neuroepidemiology Branch, NINDS Terry Phillips Ph.D. Division of Bioengineering and Physical Science, NIH, Bethesda, MD.